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The histone chaperone protein Nucleosome Assembly Protein-1 (hNAP-1) binds HIV-1 Tat and promotes viral transcription.

Vardabasso C, Manganaro L, Lusic M, Marcello A, Giacca M - Retrovirology (2008)

Bottom Line: Overexpression of hNAP-1 significantly enhanced Tat-mediated activation of the LTR.Conversely, silencing of the protein decreased viral promoter activity.Our study reveals that HIV-1 Tat binds the histone chaperone hNAP-1 both in vitro and in vivo and shows that this interaction participates in the regulation of Tat-mediated activation of viral gene expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy. vardabas@icgeb.org

ABSTRACT

Background: Despite the large amount of data available on the molecular mechanisms that regulate HIV-1 transcription, crucial information is still lacking about the interplay between chromatin conformation and the events that regulate initiation and elongation of viral transcription. During transcriptional activation, histone acetyltransferases and ATP-dependent chromatin remodeling complexes cooperate with histone chaperones in altering chromatin structure. In particular, human Nucleosome Assembly Protein-1 (hNAP-1) is known to act as a histone chaperone that shuttles histones H2A/H2B into the nucleus, assembles nucleosomes and promotes chromatin fluidity, thereby affecting transcription of several cellular genes.

Results: Using a proteomic screening, we identified hNAP-1 as a novel cellular protein interacting with HIV-1 Tat. We observed that Tat specifically binds hNAP1, but not other members of the same family of factors. Binding between the two proteins required the integrity of the basic domain of Tat and of two separable domains of hNAP-1 (aa 162-290 and 290-391). Overexpression of hNAP-1 significantly enhanced Tat-mediated activation of the LTR. Conversely, silencing of the protein decreased viral promoter activity. To explore the effects of hNAP-1 on viral infection, a reporter HIV-1 virus was used to infect cells in which hNAP-1 had been either overexpressed or knocked-down. Consistent with the gene expression results, these two treatments were found to increase and inhibit viral infection, respectively. Finally, we also observed that the overexpression of p300, a known co-activator of both Tat and hNAP-1, enhanced hNAP-1-mediated transcriptional activation as well as its interaction with Tat.

Conclusion: Our study reveals that HIV-1 Tat binds the histone chaperone hNAP-1 both in vitro and in vivo and shows that this interaction participates in the regulation of Tat-mediated activation of viral gene expression.

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Related in: MedlinePlus

hNAP-1 cooperates with Tat in LTR transactivation. A. hNAP-1 synergizes with Tat in transcriptional activation. HeLa cells were cotransfected with a reporter construct containing the HIV-1 LTR upstream of the luciferase gene, and with vectors for HA-tagged hNAP-1 (100 ng) and HIV-1 Tat (5 and 25 ng), as indicated. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The co-expression of hNAP-1 significantly increased Tat transactivation of the LTR promoter. The western blot at the bottom shows the levels of transfected hNAP-1 protein in a representative experiment. B. hNAP-1 knock down decreases Tat transactivation. HeLa cells were transfected with a specific siRNA against hNAP-1 or a control siRNA, and then transfected with the LTR-luciferase reporter together with Tat (5 and 25 ng). The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The western blot at the bottom shows the levels of endogenous hNAP-1 protein and of tubulin as a control in a representative experiment. C. hNAP-1, Tat and the acetyltransferase p300 synergistically activate viral transcription. HeLa cells were transfected with LTR-luciferase reporter plasmid and with vectors for HIV-1 Tat (5 ng), HA-hNAP-1 (100 ng) and p300 (100 ng), as indicated. After 24 h from transfection, luciferase assays were performed. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. D. p300 enhances Tat-hNAP-1 interaction in vivo. The plasmids indicated on top of the figure were transfected into HEK 293T cells. The upper panel shows western blots with the indicated antibodies after immunoprecipitation using an anti-Flag antibody; the lower three panels show western blotting controls from whole cell lysates (WCL) from transfected cells to show the levels of expression of the transfected proteins.
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Figure 4: hNAP-1 cooperates with Tat in LTR transactivation. A. hNAP-1 synergizes with Tat in transcriptional activation. HeLa cells were cotransfected with a reporter construct containing the HIV-1 LTR upstream of the luciferase gene, and with vectors for HA-tagged hNAP-1 (100 ng) and HIV-1 Tat (5 and 25 ng), as indicated. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The co-expression of hNAP-1 significantly increased Tat transactivation of the LTR promoter. The western blot at the bottom shows the levels of transfected hNAP-1 protein in a representative experiment. B. hNAP-1 knock down decreases Tat transactivation. HeLa cells were transfected with a specific siRNA against hNAP-1 or a control siRNA, and then transfected with the LTR-luciferase reporter together with Tat (5 and 25 ng). The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The western blot at the bottom shows the levels of endogenous hNAP-1 protein and of tubulin as a control in a representative experiment. C. hNAP-1, Tat and the acetyltransferase p300 synergistically activate viral transcription. HeLa cells were transfected with LTR-luciferase reporter plasmid and with vectors for HIV-1 Tat (5 ng), HA-hNAP-1 (100 ng) and p300 (100 ng), as indicated. After 24 h from transfection, luciferase assays were performed. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. D. p300 enhances Tat-hNAP-1 interaction in vivo. The plasmids indicated on top of the figure were transfected into HEK 293T cells. The upper panel shows western blots with the indicated antibodies after immunoprecipitation using an anti-Flag antibody; the lower three panels show western blotting controls from whole cell lysates (WCL) from transfected cells to show the levels of expression of the transfected proteins.

Mentions: One of the essential molecular events that parallel Tat-driven transcriptional activation is the modification of chromatin structure at the HIV-1 promoter [34,39]. We therefore investigated whether NAP-1 might contribute to Tat transactivation. A reporter construct containing the U3 and R sequences of the HIV-1 LTR upstream of the luciferase gene was co-transfected into HeLa cells, together with vectors for HA-tagged hNAP-1 and HIV-1 Tat. As shown in Figure 4A, hNAP-1, when co-transfected with Tat, significantly enhanced Tat-mediated transactivation of the LTR; hNAP-1 alone had no effect on promoter activity.


The histone chaperone protein Nucleosome Assembly Protein-1 (hNAP-1) binds HIV-1 Tat and promotes viral transcription.

Vardabasso C, Manganaro L, Lusic M, Marcello A, Giacca M - Retrovirology (2008)

hNAP-1 cooperates with Tat in LTR transactivation. A. hNAP-1 synergizes with Tat in transcriptional activation. HeLa cells were cotransfected with a reporter construct containing the HIV-1 LTR upstream of the luciferase gene, and with vectors for HA-tagged hNAP-1 (100 ng) and HIV-1 Tat (5 and 25 ng), as indicated. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The co-expression of hNAP-1 significantly increased Tat transactivation of the LTR promoter. The western blot at the bottom shows the levels of transfected hNAP-1 protein in a representative experiment. B. hNAP-1 knock down decreases Tat transactivation. HeLa cells were transfected with a specific siRNA against hNAP-1 or a control siRNA, and then transfected with the LTR-luciferase reporter together with Tat (5 and 25 ng). The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The western blot at the bottom shows the levels of endogenous hNAP-1 protein and of tubulin as a control in a representative experiment. C. hNAP-1, Tat and the acetyltransferase p300 synergistically activate viral transcription. HeLa cells were transfected with LTR-luciferase reporter plasmid and with vectors for HIV-1 Tat (5 ng), HA-hNAP-1 (100 ng) and p300 (100 ng), as indicated. After 24 h from transfection, luciferase assays were performed. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. D. p300 enhances Tat-hNAP-1 interaction in vivo. The plasmids indicated on top of the figure were transfected into HEK 293T cells. The upper panel shows western blots with the indicated antibodies after immunoprecipitation using an anti-Flag antibody; the lower three panels show western blotting controls from whole cell lysates (WCL) from transfected cells to show the levels of expression of the transfected proteins.
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Figure 4: hNAP-1 cooperates with Tat in LTR transactivation. A. hNAP-1 synergizes with Tat in transcriptional activation. HeLa cells were cotransfected with a reporter construct containing the HIV-1 LTR upstream of the luciferase gene, and with vectors for HA-tagged hNAP-1 (100 ng) and HIV-1 Tat (5 and 25 ng), as indicated. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The co-expression of hNAP-1 significantly increased Tat transactivation of the LTR promoter. The western blot at the bottom shows the levels of transfected hNAP-1 protein in a representative experiment. B. hNAP-1 knock down decreases Tat transactivation. HeLa cells were transfected with a specific siRNA against hNAP-1 or a control siRNA, and then transfected with the LTR-luciferase reporter together with Tat (5 and 25 ng). The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The western blot at the bottom shows the levels of endogenous hNAP-1 protein and of tubulin as a control in a representative experiment. C. hNAP-1, Tat and the acetyltransferase p300 synergistically activate viral transcription. HeLa cells were transfected with LTR-luciferase reporter plasmid and with vectors for HIV-1 Tat (5 ng), HA-hNAP-1 (100 ng) and p300 (100 ng), as indicated. After 24 h from transfection, luciferase assays were performed. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. D. p300 enhances Tat-hNAP-1 interaction in vivo. The plasmids indicated on top of the figure were transfected into HEK 293T cells. The upper panel shows western blots with the indicated antibodies after immunoprecipitation using an anti-Flag antibody; the lower three panels show western blotting controls from whole cell lysates (WCL) from transfected cells to show the levels of expression of the transfected proteins.
Mentions: One of the essential molecular events that parallel Tat-driven transcriptional activation is the modification of chromatin structure at the HIV-1 promoter [34,39]. We therefore investigated whether NAP-1 might contribute to Tat transactivation. A reporter construct containing the U3 and R sequences of the HIV-1 LTR upstream of the luciferase gene was co-transfected into HeLa cells, together with vectors for HA-tagged hNAP-1 and HIV-1 Tat. As shown in Figure 4A, hNAP-1, when co-transfected with Tat, significantly enhanced Tat-mediated transactivation of the LTR; hNAP-1 alone had no effect on promoter activity.

Bottom Line: Overexpression of hNAP-1 significantly enhanced Tat-mediated activation of the LTR.Conversely, silencing of the protein decreased viral promoter activity.Our study reveals that HIV-1 Tat binds the histone chaperone hNAP-1 both in vitro and in vivo and shows that this interaction participates in the regulation of Tat-mediated activation of viral gene expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy. vardabas@icgeb.org

ABSTRACT

Background: Despite the large amount of data available on the molecular mechanisms that regulate HIV-1 transcription, crucial information is still lacking about the interplay between chromatin conformation and the events that regulate initiation and elongation of viral transcription. During transcriptional activation, histone acetyltransferases and ATP-dependent chromatin remodeling complexes cooperate with histone chaperones in altering chromatin structure. In particular, human Nucleosome Assembly Protein-1 (hNAP-1) is known to act as a histone chaperone that shuttles histones H2A/H2B into the nucleus, assembles nucleosomes and promotes chromatin fluidity, thereby affecting transcription of several cellular genes.

Results: Using a proteomic screening, we identified hNAP-1 as a novel cellular protein interacting with HIV-1 Tat. We observed that Tat specifically binds hNAP1, but not other members of the same family of factors. Binding between the two proteins required the integrity of the basic domain of Tat and of two separable domains of hNAP-1 (aa 162-290 and 290-391). Overexpression of hNAP-1 significantly enhanced Tat-mediated activation of the LTR. Conversely, silencing of the protein decreased viral promoter activity. To explore the effects of hNAP-1 on viral infection, a reporter HIV-1 virus was used to infect cells in which hNAP-1 had been either overexpressed or knocked-down. Consistent with the gene expression results, these two treatments were found to increase and inhibit viral infection, respectively. Finally, we also observed that the overexpression of p300, a known co-activator of both Tat and hNAP-1, enhanced hNAP-1-mediated transcriptional activation as well as its interaction with Tat.

Conclusion: Our study reveals that HIV-1 Tat binds the histone chaperone hNAP-1 both in vitro and in vivo and shows that this interaction participates in the regulation of Tat-mediated activation of viral gene expression.

Show MeSH
Related in: MedlinePlus